PROJECT SUMMARY Nucleosomes, the basic repeating unit of chromatin, are comprised of DNA wrapped around a histone octamer core. Histones are decorated with a variety of post-translational modifications (PTMs), which interact with proteins to regulate diverse biological processes and disease. PTMs are a rapidly growing target in biomedical research, and are typically analyzed via antibody-dependent chromatin immunoprecipitation (ChIP) and enzyme-linked immunosorbent assays (ELISAs). Thus, the generation of highly specific antibodies is a top priority for the field, both for academic research, and for its application to diagnostic / prognostic assays and therapeutics. EpiCypher pioneered the commercialization of recombinant designer nucleosomes (dNucs) for PTM antibody specificity testing with our SNAP-ChIP (Sample Normalization and Antibody Profiling) platform. Using SNAP-ChIP, we found that >80% of commercially available PTM antibodies display a striking amount of cross- reactivity. Such widespread deficiencies are partly due to histone PTM antibody development protocols, which rely on histone peptides for immunization, selection and validation. We found that histone peptide arrays fail to generate reliable antibody specificity profiles, and that a nucleosome-based substrate, representative of the in vivo physiological context, is required for predicting PTM antibody performance in native chromatin. However, the size of conventional immunoglobulins (~150kDa) has also made it difficult to target PTMs in or near the histone globular domain, adding another layer of complexity to PTM antibody development. Clearly, new technologies are required to generate highly specific reagents capable of defining PTMs in clinical assays. Here, EpiCypher will develop recombinant Nucleobodies? (Nucleosome-binding single-domain antibodies) to PTMs that have been challenging to target with conventional methods (H3K79me2 and H3K4me3). Single-domain antibodies (sdAbs) are derived from camelid (e.g. llama) heavy-chain-only antibodies (HcAbs). sdAbs are small (~15kDa), soluble / stable in solution, and fully sequenced, making them highly diverse, renewable tools. The innovation of our proposal is the development of highly specific sdAbs to PTMs (Nucleobodies), utilizing dNucs for immunization and as reliable physiological substrates for candidate selection and final functional validation. In Aim 1 of this proposal, we will develop Nucleobodies to H3K79me2 and H3K4me3, using dNucs for immunization and on- and off-target screening. In Aim 2, we will identify ChIP- grade Nucleobodies with our proprietary SNAP-ChIP technology, and validate reagents in quantitative ELISA and ChIP-seq, establishing the utility of these tools in chromatin research and clinically relevant applications. Nucleobodies have the potential to dramatically expand the variety of PTMs available for biomedical research, and with their increased stability / specificity, will accelerate the application of PTMs as biomarkers and drug targets.